Ann Kaminski

Mechanism of Initiation Site Selection Promoted by the Human Rhinovirus 2 Internal Ribosome Entry Site

ABSTRACT Translation initiation site usage on the human rhinovirus 2 internal ribosome entry site (IRES) has been examined in a mixed reticulocyte lysate/HeLa cell extract system. There are two relevant AUG triplets, both in a base-paired hairpin structure (domain VI), with one on the 5′ side at nucleotide (nt) 576, base paired with the other at nt 611, which is the initiation site for polyprotein synthesis. A single residue was inserted in the apical loop to put AUG-576 in frame with AUG-611, and in addition another in-frame AUG was introduced at nt 593. When most of the IRES was deleted to generate a monocistronic mRNA, the use of these AUGs conformed to the scanning ribosome model: improving the AUG-576 context increased initiation at this site and decreased initiation at downstream sites, whereas the converse was seen when AUG-576 was mutated to GUA; and AUG-593, when present, took complete precedence over AUG-611. Under IRES-dependent conditions, by contrast, much less initiation occurred at AUG-576 than in a monocistronic mRNA with the same AUG-576 context, mutation of AUG-576 decreased initiation at downstream sites by ∼70%, and introduction of AUG-593 did not completely abrogate initiation at AUG-611, unless the apical base pairing in domain VI was destroyed by point mutations. These results indicate that ribosomes first bind at the AUG-576 site, but instead of initiating there, most of them are transferred to AUG-611, the majority by strictly linear scanning and a substantial minority by direct transfer, which is possibly facilitated by the occasional persistence of base pairing in the apical part of the domain VI stem.

The RNA-binding properties of protein synthesis initiation factor eIF-2.

Protein synthesis initiation factor eIF-2 bound ATP in the presence or absence of Mg2+ ions. ATP impaired the binding of GTP or GDP to eIF-2. However, excess GTP did not significantly decrease the binding of ATP to eIF-2, suggesting eIF-2 has distinct ATP and GTP binding sites. Highly purified eIF-2 can bind mRNA, and this did not require the mRNA to be capped. mRNA binding was saturable, and maximal binding corresponded to about 0.4 mol mRNA bound per mol eIF-2. GTP, and, at lower concentrations, GDP, inhibited the binding of mRNA to eIF-2. In addition, ATP and other nucleoside triphosphates decreased mRNA binding. The implications of these findings for the structure and function of eIF-2 are discussed. Preparations of eIF-2 deficient in the beta-subunit showed reduced ability to bind mRNA, suggesting that while it is not essential for mRNA binding, this subunit is involved in the interaction. Consistent with this is the observation that ultraviolet crosslinking of mRNA to eIF-2 resulted primarily in labelling of the beta-subunit. Subsequent analysis revealed that mRNA was cross-linked to the C-terminal region of eIF-2b which contains a putative Zn-finger structure.

8 Coupled Termination-Reinitiation Events in mRNA Translation

Bicistronic (and polycistronic) mRNA translation is commonplace in eubacteria, although translation of the downstream cistrons may not invariably involve coupled termination-reinitiation events. In contrast, efficient translation of the downstream cistron of a bicistronic mRNA is exceedingly rare in eukaryotes, unless there is an internal ribosome entry site (IRES) or unless the upstream cistron is very short. In this chapter, we first review the eubacterial mechanism of coupled termination-reinitiation and then discuss the reasons that similar events do not generally occur in eukaryotes. We then review the mechanism underlying one of the few exceptional cases of reinitiation on a eukaryotic bicistronic mRNA with a long upstream open reading frame (ORF), and we conclude by examining what it is that is so very different when the ORF is short that it results in reinitiation being the normal (default) outcome. TRANSLATIONAL COUPLING AND TERMINATION-REINITIATION IN PROKARYOTES Each cistron of a eubacterial polycistronic mRNA usually has its own Shine-Dalgarno (SD) motif, which should, in principle, allow it to be translated independently. However, in a great many polycistronic mRNAs, there is translational coupling: Initiation of translation of a downstream cistron (cistron n + 1) is quite strictly dependent on translation of an upstream cistron, generally the immediate upstream cistron (cistro n n ). The usual explanation is that the initiation site of cistron n + 1 is occluded by secondary structure base-pairing with the coding region of cistron n (often the far upstream coding region), and so it requires translation of cistron n to unwind this secondary...